Measurement of the luminescence emission of substances has become increasingly important as a sensitive analytical technique in a wide range of disciplines such as chemistry, biology, engineering, materials science and application areas ranging from pollution monitoring and production control in a chemical plant to the sequencing of DNA. In many of these areas, distinguishing the weak but characteristic emission of the substance of interest from background light is difficult. Measuring the lifetime of the light emission of the substance of interest can often be used to discriminate the desired signal from a background. In this context we define the luminescence lifetime of a substance to be the time taken for the intensity of light emitted in response to an exciting pulse of light to fall to 1/e (˜1/2.7) of its initial value. This characteristic time can range from less than one nanosecond to minutes, depending on the processes involved. Such processes can be described as fluorescence, luminescence or phosphorescence, but if the desired light emission has a lifetime which is different from light of another source, its detection can be enhanced.
One example of fluorescence measurements is in DNA sequencing, where molecular fragments are ‘labelled’ with a fluorescent dye and the fluorescence emission of a dye-labelled fragment is used to identify which amino acid base begins or ends a given sequence of amino acids. Another application is in the screening of potential drugs against a target molecule. Typically changes in fluorescence intensity are used to identify ‘hits’ in response to a chemical test or ‘assay’ of the interaction between a potential drug and a target molecule. In this case, either the drug or the target or both may be labelled. In both sequencing and screening, the characteristic lifetime of a fluorescent molecule can be used to improve its detection.
A fluorescent material is a material which can be excited to a higher electronic state and on relaxing towards its ground state emits a photon. The time elapsed from the excitation until the emission of a photon, the intensity of emitted photons, and the energy (frequency) of the photons are all properties that can be measured and used to characterize the material. The excited state is typically achieved by subjecting the material to a short light pulse. The emission of photons is a Poisson process and the intensity of the emitted photons is continuously decreasing, and typically follows an exponential decay. A useful way of characterising a fluorescent material is to measure the time elapsed between an excitation and the arrival of photons at a detector. λ(t) denotes the Poisson arrival rate at time t at all times and is given by:λ(t)=Ae−αt  equation 1where A denotes the intensity of a single fluorescent material, measured in photons per second, at time 0 (the time of the excitation), also referred to as the initial intensity, α denotes the decay constant of the fluorescence, measured in nepers/second, and 1/α is the fluorescence lifetime, which is often used to characterise a fluorescent material. If a sample has more than one fluorescent material present, the arrival rate of photons can typically be described by a multiexponential expression.
A widely used technique of determining the fluorescence lifetime of a material is Time Correlated Single Photon Counting (TCSPC). In TCSPC a sample is typically subjected to a short light pulse, which excites the material in the sample. The material will, if fluorescent, emit a photon a short time after the excitation. The emitted photon is detected and the time that has elapsed between the excitation light pulse and the arrival of the first emitted fluorescence photon is recorded by the measurement system. The technique is only capable of counting a maximum of one photon per excitation. In practice less than one photon per excitation are counted since, to get a representative distribution of arrival times, a low intensity of the exciting light pulse is used, giving a low probability of photon emission. Therefore, to get a reliable measure of the fluorescence lifetime characteristic of a particular material, using TCSPC, i.e. to acquire a fluorescence decay curve, the process of exciting the material in the sample and detecting the first emitted photon, has to be repeated a large number of times. A measurement can typically involve several tens of thousands excitation-photon detection cycles. The TCSPC method has been continuously improved, for example U.S. Pat. No. 5,990,484 teaches a method and apparatus for counting a total number of emitted photons, allowing a higher intensity of the exciting light pulse, and U.S. Pat. No. 4,686,371 teaches a method of compensating for variations in the length and intensity of the exciting light pulse.
As above indicated, TCSPC can, even if the mentioned improvements are utilised, be a time consuming measurement. It is realised in the art that the method is wasteful in that only the first emitted photon is detected and used for the determination of the fluorescence lifetime, even though the subsequently emitted photons carry information useful for the determination of the characteristic lifetime. A number of approaches have been suggested to extend the TCSPC to also detect and record subsequent photons. The most recent development of the TCSPC-technique includes various multiplexing techniques, in which, in principle, a plurality of detectors are connected to one or more analysing means, examples described in Multiplexed single-photon counting by Scubling et al., Rev. Sci. Instrum. 67 (6), 2238-2246 June 1996. Alternatively multi-element detectors have been suggested, for example by Browne et al. in A 100 ns anti-coincidence circuit for multi-channel counting systems, Meas. Sci. Technol. 6 (6), 1487-1491, June 1996. The suggested Multiplexed Time Correlated Single-Photon Counting (M-TCSPC)—techniques represent significant improvements over the TCSPC—techniques, but have the drawback that the number of photons that it is possible to detect following one excitation pulse is limited by the number of detectors (or detector elements) used; therefore a large number of excitation cycles is still needed to determine the fluorescence lifetime of the material. In addition multielement detectors are costly, as are systems providing a large number of single detectors in a multiplexing arrangement.
The known apparatus and measurement techniques thus suffer, as above described, from a number of disadvantages and limitations, the most important being the long measurement time. Thus, there is a need in the art to provide a measurement system and method that shortens measurement time.